Extracellular Matrix—Key to Maintaining Function of Encapsulated Human Stem Cell Differentiated Islet Clusters Seeded into Scaffolds as a Diabetes Therapy

Tissue ischemia promotes vasculogenesis through chemokine-induced recruitment of bone marrow-derived endothelial progenitor cells (EPCs). Diabetes significantly impairs this process. Because hyperglycemia increases reactive oxygen species in a number of cell types, and because many of the defects responsible for impaired vasculogenesis involve HIF1-regulated genes, we hypothesized that HIF1 function is impaired in diabetes because of reactive oxygen species-induced modification of HIF1alpha by the glyoxalase 1 (GLO1) substrate methylglyoxal. Decreasing superoxide in diabetic mice by either transgenic expression of manganese superoxide dismutase or by administration of an superoxide dismutase mimetic corrected post-ischemic defects in neovascularization, oxygen delivery, and chemokine expression, and normalized tissue survival. In hypoxic fibroblasts cultured in high glucose, overexpression of GLO1 prevented reduced expression of both the EPC mobilizing chemokine stromal cell-derived factor-1 (SDF-1) and of vascular epidermal growth factor, which modulates growth and differentiation of recruited EPCs. In hypoxic EPCs cultured in high glucose, overexpression of GLO1 prevented reduced expression of both the SDF-1 receptor CXCR4, and endothelial nitric-oxide synthase, an enzyme essential for EPC mobilization. HIF1alpha modification by methylglyoxal reduced heterodimer formation and HIF1alpha binding to all relevant promoters. These results provide a basis for the rational design of new therapeutics to normalize impaired ischemia-induced vasculogenesis in patients with diabetes.

Tannic Acid Capsules Decrease Redox-dependent Pro-inflammatory Responses to Prolong Islet Graft Survival After Transplantation

During bone formation, osteoblasts deposit an extracellular matrix (ECM) that is mineralized via a process involving production and secretion of highly specialized matrix vesicles (MVs). Activin A, a transforming growth factor-β (TGF-β) superfamily member, was previously shown to have inhibitory effects in human bone formation models through unclear mechanisms. We investigated these mechanisms elicited by activin A during in vitro osteogenic differentiation of human mesenchymal stem cells (hMSC). Activin A inhibition of ECM mineralization coincided with a strong decline in alkaline phosphatase (ALP(1)) activity in extracellular compartments, ECM and matrix vesicles. SILAC-based quantitative proteomics disclosed intricate protein composition alterations in the activin A ECM, including changed expression of collagen XII, osteonectin and several cytoskeleton-binding proteins. Moreover, in activin A osteoblasts matrix vesicle production was deficient containing very low expression of annexin proteins. ECM enhanced human mesenchymal stem cell osteogenic development and mineralization. This osteogenic enhancement was significantly decreased when human mesenchymal stem cells were cultured on ECM produced under activin A treatment. These findings demonstrate that activin A targets the ECM maturation phase of osteoblast differentiation resulting ultimately in the inhibition of mineralization. ECM proteins modulated by activin A are not only determinant for bone mineralization but also possess osteoinductive properties that are relevant for bone tissue regeneration.

Catalase overexpression attenuates angiotensinogen expression and apoptosis in diabetic mice

Emergent technologies in regenerative medicine may soon overcome the limitations of conventional diabetes therapies. Collaborative efforts across the subfields of stem cell technology, islet encapsulation, and biomaterial carriers seek to produce a bioengineered pancreas capable of restoring endocrine function in patients with insulin-dependent diabetes. These technologies rely on a robust understanding of the extracellular matrix (ECM), the supportive 3-dimensional network of proteins necessary for cellular attachment, proliferation, and differentiation. Although these functions can be partially approximated by biosynthetic carriers, novel decellularization protocols have allowed researchers to discover the advantages afforded by the native pancreatic ECM. The native ECM has proven to be an optimal platform for recellularization and whole-organ pancreas bioengineering, an exciting new field with the potential to resolve the dire shortage of transplantable organs. This review seeks to contextualize recent findings, discuss current research goals, and identify future challenges of regenerative medicine as it applies to diabetes management.

Fractalkine (FKN) is a unique chemokine that works as a chemoattractant and an adhesion molecule. Previous studies have demonstrated that FKN plays a role in ischaemic and protein-overload renal injury via its cognate receptor chemokine (C-X3-C motif) receptor 1 (CX3CR1). However, involvement of the FKN/CX3CR1 system in diabetic nephropathy remains unclear. We examined the role of FKN/CX3CR1 in diabetic mice and mouse mesangial cells (MMCs). Streptozotocin (50mgkg(-1)day(-1)) was intraperitoneally administered for 5days to male Cx3cr1-knockout (KO) mice and wild-type (WT) mice. MMCs transfected with Fkn (also known as Cx3cl1) or Cx3cr1 siRNA, respectively, were used to elucidate the role of FKN/CX3CR1 in extracellular matrix (ECM) synthesis. At 12weeks, diabetic Cx3cr1 KO mice showed no significant changes in plasma glucose, but markers of renal inflammation, fibrosis and ECM, such as the fractional mesangial area, fibronectin and collagen, were significantly lower in diabetic Cx3cr1 KO mice compared with diabetic WT mice. High glucose, oleic acid and TGF-β1 stimulated FKN and CX3CR1 expression, together with the expression of ECM proteins in MMCs, but the effects were significantly attenuated by Fkn or Cx3cr1 siRNA. More importantly, FKN itself increased mesangial ECM through CX3CR1 and subsequent activation of reactive oxygen species and mitogen-activated protein kinases. A neutralising TGF-β antibody inhibited FKN/CX3CR1 in MMCs treated with diabetic stimuli and decreased FKN-induced ECM accumulation. These results demonstrate that FKN/CX3CR1 may play an important role in diabetic renal injury through upregulation of ECM synthesis and could therefore be a therapeutic target for preventing diabetic nephropathy.

Prunella vulgaris L. (Labiatae) has been reported to have a wide range of health benefits in oriental medicine. This study for the first time is to examine the antihyperglycemic effects of P. vulgaris in streptozotocin (STZ)-induced diabetic ICR mice (STZ diabetic mice). The effects of P. vulgaris L. aqueous-ethanol extract (PVE) on blood glucose, exogenous insulin sensitivity and plasma insulin levels were investigated. In four doses of extracts from the spikes of P. vulgaris, extract at dose of 100 mg/kg significantly suppressed the rise in blood glucose after 30 min in the acute glucose tolerance test. Furthermore, this dose was applied in the fellow experiments. A significant decrease in blood glucose levels was observed after treatment of PVE. A combination of PVE and glibenclamide produced a greater effect in blood glucose level than using glibenclamide or PVE alone. PVE enhanced and prolonged the antihyperglycemic effects of exogenous insulin on STZ diabetic mice. Plasma insulin levels were increased with glibenclamide treatment in STZ diabetic mice, whereas such effect was not observed with PVE. These results indicated that P. vulgaris enhances the antihyperglycemic effects of exogenous insulin without stimulating insulin secretion, indicating that insulin sensitivity is increased in STZ diabetic mice.

Mitochondrial (mito) bioenergetics of circulating cells change as a function of disease in Alzheimer’s, fibromyalgia, cancer, and Type 2 diabetes. We are exploring the use of mito-bioenergetics as a biomarker of Type 1 diabetes (T1D) progression and immune cell activation. T1D is a polygenic autoimmune disease in which an inflammatory leukocytic infiltrate into pancreatic islets results in destruction of insulin-producing β-cells. Autoantibody synthesis is currently used as a biomarker, but is a poor predictor of T1D since the majority of β-cells are destroyed prior to autoantibody seroconversion. We hypothesize that T1D is a chronic inflammatory disease in which conditions of oxidative stress will diminish the mitochondrial reserve capacity of immune cells and provide a surrogate marker for T1D. We compared mito-bioenergetics of murine immune cells derived from the spleen, pancreatic lymph node and bone marrow between diabetic and pre-diabetic Non-Obese Diabetic (NOD) mice. We show that mitochondrial reserve capacity is reduced in circulating CD4+ T cells from diabetic mice, but not in pre-diabetic mice during T1D progression. To explore the role of mitochondrial oxidative stress on diabetogenic CD4+ T cell effector responses, we used mito-TEMPO to scavenge mitochondrial reactive oxygen species (mROS). Mito-TEMPO reduced IFNγ synthesis in a primary recall assay by 0.24-fold and the combination of mito and cytosolic TEMPO increased Treg-associated IL-2 by 2.5-fold, suggesting that mROS have a causal role in modulating T cell effector responses critical for T1D pathology. We plan to perform human translational studies on mito-bioenergetics of PBMCs from recent onset T1D patients and further dissect the role of mROS on T1D.

Robot-assisted orthotopic and heterotopic ovarian tissue transplantation techniques: surgical advances since our first success in 2000

The Official Journal of ATTD Advanced Technologies &amp; Treatments for Diabetes Conference <i>Berlin, Germany—February 20–23, 2019</i>

Islet Encapsulation: A Long-Term Treatment for Type 1 Diabetes

Quiescence regulators for hematopoietic stem cell

We have previously shown that mice and human patients with diabetes mellitus develop microangiopathy in bone marrow (BM) resulting in depletion of stem cells (SCs). Since the endosteal niche was the most pauperised, we decided to investigate if activation of osteoclasts (OCLs) may participate in this process through detachment of SC from the endosteal niche leading to inappropriate mobilization. The study was performed in streptozotocin-induced type 1 diabetic (T1D) mice at 5, 11 and 20 weeks from induction of diabetes and in age-matched non-diabetic controls (C). Histochemical analysis of femoral bones at late stages of DM (20 weeks) showed a profound BM remodeling characterized by reduction of the hematopoietic component, fat accumulation and bone rarefaction. The activation of osteoclastogenesis was already seen at 5 weeks as assessed by counting the number of tartrate resistant acid phosphatase (TRAP) positive OCLs in the trabecular bone (2.9±0.3 vs. 1.6±0.7 cells/mm of endosteal length in C, p<0.01). Osteoclast activation was associated with a marked increase in peripheral blood lineage- Sca1+ cKit+ (LSK) cells (0.7±0.4 vs. 0.2±0.1% of total MNCs in C, p<0.05). Unexpectedly, mRNA and protein expression of Receptor Activator of NF-κB Ligand (RANKL), an inducer of OCL differentiation and activation, was reduced in total BM at 20 and 11 weeks (mRNA: 0.3±0.01 vs. 1.2±0.3 ddCT in C; Protein: 11.8±2 vs. 28.7±5 pg/ml, p<0.05 for both comparisons) thus implying that OCL activation occurs through a non-canonical mechanism. Interestingly, we found T1D induces acidosis in BM (pH: 6.9±0.02 vs. 7.0±0.03 in C, p<0.05). Moreover, similar pH changes in culturing media increased the number of BM-derived OCL-like cells in vitro (2.0±0.3 fold increase relative to C, p<0.05) suggesting a crucial role of acidosis in OCLs activation. In summary, parallel increases in circulating SCs and endosteal OCLs occur at initial phase of T1D, before any apparent alteration of hematopoiesis. OCL activation may lead to detachment of SCs from the endosteal niche and excessive mobilization, thus resulting in BM exhaustion at later stages. OCL-induced remodeling of the endosteal niche, which contains the most primitive SCs in BM, may contribute to the impairment of regenerative capacity in T1D.

News and issues that affect organ and tissue transplantation

Type 1 diabetes (T1D) is a chronic condition in which patients suffer from high blood glucose levels due to the body's inability to produce sufficient insulin. Continuous insulin administration and T1D management are difficult, often leading to hypoglycemic events, insulin resistance, and lower quality of life. Major advancements have been made in recent years, including clinical islet transplantation, but their application is limited by rapid immune rejection and islet destruction. Thus, a necessary paradigm shift has been observed in recent times toward biomaterial-based islet transplantation therapy. The use of biomaterial-based encapsulation addresses major limitations, including immune rejection and hypoxia, and provides a proper cell microenvironment offering greater islet viability. Presently, researchers are more focused on developing a clinically translatable therapy for T1D with the existing knowledge of advanced biomaterial technology. In this review article, we provide a historical perspective, highlighting the developments in the field of islet encapsulation and transplantation, and focus on cutting-edge advancements with modern bioengineering from a clinical perspective.